Zebrafish maintenance and breeding

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SIRT1 in Zebrafish

There is very little published research on the function of SIRT1 in zebrafish even though zebrafish have been shown to be a good model for studying SIRT1 (Pereira et al. 2011). It has been showed in zebrafish that SIRT1 acts as a regulator in endothelial angiogenesis and is essential for normal postnatal vascular growth (Guarani et al. 2011; Potente et al. 2007). Transcription levels of sirt1 mRNA have been showed to be reduced in the brain of zebrafish when the fish are exposed to hypoxia and it has thus been suggested that SIRT1 is involved in the brain response to oxygen scarcity (Zakhary et al. 2011). Hypoxia has also been shown to affect the sex ratio of zebrafish, increasing the percentage of males (Shang et al. 2006), showing a possible pathway for SIRT1 to affect sex ratio (Figure 3).
Figure 3. Possible link between SIRT1 and male sex ratio. Hypoxia has been shown to cause an increase of male sex ratio thru increased apoptosis (Shang et al. 2006). It has also been showed in zebrafish brain cells that hypoxia leads to a reduction of sirt1 mRNA levels (Zakhary et al. 2011). Down-regulation of SIRT1 has been showed to an increase in apoptosis.

EX-527

Indole EX-527 (6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide9) is an inhibitor of SIRT1 that has a 100-fold higher affinity towards SIRT1 than any of the other sirtuins (Napper et al. 2005). The mechanism of inhibition is not fully understood. However, it has been suggested that it is achieved by EX-527 binding to NAD+ and two pockets in the catalytic domain of the SIRT1 protein, thereby preventing the release of the enzymatic coproduct 2’-O-acetyl-ADP ribose and thus inhibiting further enzymatic reactions (Gertz et al. 2013).

Zebrafish sex differentiation

Sex differentiation in zebrafish remains elucive. Male zebrafish follow the same development path as females during the early stage of gonad differentiation, developing juvenile ovaries (Takahashi 1977; Wang et al. 2007). In males around 15–19 days post fertilisation (dpf) the juvenile ovaries start to undergo apoptosis and transform into testes whereas in females they continue to develop into mature ovaries (Pradhan et al. 2012; Uchida et al. 2002). Blocking apoptosis in the juvenile ovaries through activation of NFκB has been shown to yield female biased populations (Pradhan et al. 2012).
Mammals have sex chromosomes that determine the sex through the presence or absence of the sex determening box y (sry) gene. No such sex chromosomes or master regulator of the sex-genes have been reported in zebrafish (Sekido and Lovell-Badge 2008; Traut and Winking 2001; Wallace and Wallace 2003). The genes downstream of Sry in mammals are conserved in zebrafish and function in similar ways as in mammals (Orban et al. 2009; Rodriguez-Mari et al. 2005; Siegfried and Nusslein-Volhard 2008; Sreenivasan et al. 2013). Environmental factors such as hypoxia, thermal cycling and temperature have been showed to have an effect on the sex differentiation process (Ospina-Alvarez and Piferrer 2008; Shang et al. 2006; Villamizar et al. 2012). However, while these enviromental factors do effect the sex ratio they do not appear to determine the sex of the zebrafish since they do not show a total control of the sex ratio, complicating the system further.

Aim of the study

The role of SIRT1 in zebrafish (Danio rerio) is not well understood. Studying the role of SIRT1 in zebrafish may give a better understanding of the function of thee gene and reveal if it is involved in sex differentiation.
The purpose of this study was to examine if inhibition of SIRT1 would affect the sex differentiation process in zebrafish and if it would affect the expression of apoptotic and sex related genes.

MATERIAL AND METHODS

Zebrafish maintenance and breeding

For all the experiments transgenic vas::EGFP zebrafish were used (Krøvel, 2002). The transgenic zebrafish express EGFP in the female germline while in male the transcript is degraded. This makes it possible to determine sex of the zebrafish at 50 dpf.
Adult zebrafish were maintained in a recirculating system (Aquaneering, USA) with a 14-h light/10-h dark cycle. The fish were fed twice a day with newly hatched Artemia salina nauplii and commercial flake food (Tetrarubin) (Larval AP 100). Juvenile fish were fed with smaller commercial flake food (Larval AP 100). The male and female brooders were kept in separate aquaria at 25 ± 1° C and were allowed to breed once a week in mating containers (Aquaneering, USA). Collected eggs where kept at 28° C. At 4 dpf the juveniles were transferred to the circulating system. The water flow was adjusted to 20–30 drops/min, 14-h light/10-h dark cycle. The fish handling procedures were approved by the Swedish Ethical Committee in Linköping (Permit 32-10).

Molecular phylogenetic study

Protein sequences were used in the homology alignment and construction of the phylogenetic tree had the following accession numbers: NP_036370.2 for human, XP_003312628.1 for chimpanzee, NP_062786.1 for mouse, XP_003753523.1 for rat, NP_001004767.1 for chicken, NP_001091195.1 for xenopus, XP_001334440.4 for zebrafish, XP_004077552.1 for oryzias, NP_477351.1 for drosophila, NP_001255485.1 for C. elegans, XP_960372.1 for neurospora and NP_014573.1 for saccharomyces. Homology alignment of the sequences was achieved using the MEGA5 (version 5.2) software (Tamura et al. 2011).
The MEGA5 program was used to construct a phylogenetic tree using the Neighbor-Joining method to infer the phylogenetic relations between the sequences (Saitou, 1987). A bootstrap test (1000 replicates) was performed. Evolutionary distances were calculated using the p-distance method. Positions containing gaps and missing data was deleted.

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Computational modeling

Molecular modeling was performed using the Molecular Operating Environment (MOE 2012.10) software. Crystal structure of human SIRT1 catalytic domain was obtained from the Protein Data Bank (PDB). PDB identification code of the crystal structure is 4I5I. Structure preparation of 4I5I was performed before it was used as template for homology modeling of zebrafish SIRT1 catalytic domain. The Amber99 force field was used during homology modeling of the zebrafish SIRT1 catalytic domain (Summa and Levitt 2007). NAD+, EX-527 and water molecules were included in the calculation of homology.

Exposure of zebrafish

Freshly fertilized eggs were collected in the morning and transferred into 6 well plates (BD Falcon, USA) with 40 eggs in each well. They were exposed to one of the following concentrations of EX-527 (Tocris Biosciences, UK); 0.1 µM, 1 µm or 10 µM. The exposure lasted for 144 hr. The number of hatched and dead fish was counted every day. The eggs were kept at 25 ± 1° C. At 72 h post fertilization 24 samples from each well was snap-frozen in liquid nitrogen. The samples were stored in -80° C until further use. At the end of the exposure the fish were studied for morphological changes.
Fifteen days old zebrafish were divided into groups of 25 and transferred into 115-mm crystallization beakers containing 100 ml of water in which they were exposed to either 0.1 µM or 1 µm of EX-527. They were kept at 25 ± 1° C. Fifty percent of the water was changed every other day. EX-527 was added to maintain the initial concentration. After two and six days of exposure fish were collected and snap-frozen in liquid nitrogen and stored in -80° C until further use.

RNA extraction and qRT-PCR

All samples were homogenized in 400 µl of TRI-Reagent (Sigma, USA) and RNA was isolated using the manufacturer’s instruction. Synthesis of cDNA was done using qScript cDNA synthesis kit (Quanta Bioscience, USA). cDNA was stored in -20° C until further use. Primers used in qRT-PCR were designed for listed genes (Table 1). SYBR Green (KapaBiosystems, USA) was used to determine the transcription levels of all genes. Thermocycling conditions for SYBR Green were 95° C for 2 s and 60° C for 30 s. elongation factor 1 alpha 1 (ef1a/1) was used for normalizing the values. Data analysis was performed using the standard curve method (Schmittgen and Livak 2008)). qRT-PCR analysis was done for the listed genes (supplementary Table S1).

Western Blot analysis

Samples of the juvenile zebrafish exposed for two days were homogenized in radioimmune precipitation assay buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Trizma base, pH 8.0). Protein concentration was estimated using Bradford reagent (Bio-Rad). 30 µg of each sample was loaded into a 10% SDS-PAGE. The protein was then transferred to a PVDF membrane (Amersham Bioscience) using a transfer cassette and transfer buffer (25mM Tris base, 192mM Glycine, 20% Methanol). The membrane was incubated for 1 h in Tris-buffered saline containing 0.1% Tween to prevent nonspecific binding. Thereafter the membrane was probed with anti-SOX9A antibodies (AnaSpec) at 1:500 dilution and incubated at 4° C overnight. The HRP-conjugate anti-rabbit IgG (Amersham Bioscience) was used at a 1:3000 dilution for 2 h, and the immunoreactive complex was detected using Super Signal West Pico chemiluminescent substrate (Thermo Scientific). The membrane was then stripped and probed with β-actin antibody (Sigma) at 1:5000 dilution for 2 h. The HRP-conjugate anti-mouse IgG (Amersham Bioscience) was used at a 1:3000 dilution for 2 h. The bands were quantified using the ImageJ software (National Institute of Health) and normalized after respective β-actin level.

Statistical analysis

Statistical significance was determined using one way ANOVA followed by Dunnett posttest. Differences in values were considered significant if the p values were less than 0.05 (*p < 0.05, ** p < 0.01, ** p < 0.001). Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, USA).

RESULT AND DISCUSSION

The catalytic domain of SIRT1 is conserved

During the evolutionary history of SIRT1 (Figure 1) the human SIRT1 has evolved further than the zebrafish SIRT1. The zebrafish SIRT1 gene shows highest homology to other teleost genes and is evolutionary separated both from the ancestral genes of the avian and mamalian counterparts (Table 1). The catalytic core of SIRT1 has been conserved to a much higher degree than the N- and C-terminal regionas (Figure 2). A computional homology model of the catalytic domain of zebrafish SIRT1 was constructed and showed high structural similarity to the catalytic domain of human SIRT1 (Figure 4).

Table of contents :

ABSTRACT
SAMMANFATTNING
1. INTRODUCTION
1.1 SIRT1
1.2 SIRT1 in Zebrafish
1.3 EX-527
1.4 Zebrafish sex differentiation
1.5 Aim of the study
2. MATERIAL AND METHODS
2.1 Zebrafish maintenance and breeding
2.2 Molecular phylogenetic study …
2.3 Computational modeling
2.4 Exposure of zebrafish
2.5 RNA extraction and qRT-PCR
2.6 Western Blot analysis
2.7 Statistical analysis
3. RESULT AND DISCUSSION
3.1 The catalytic domain of sirt1 is conserved
3.2 Exposure to EX-527 down-regulates sex related genes in larval and juvenile zebrafish
3.3 Exposure to EX-527 up-regulates apoptotic related genes in larval and juvenile zebrafish
3.4 Exposure to EX-527 causes malformations and higher mortality
3.5 EX-527 exposure does not affect sex ratio
4. CONCLUSION
AKNOWLEDGEMENTS
REFERENCES
SUPPLEMENTARY DATA.

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